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目的利用正常人源肝细胞(HepaRG)和高内涵技术检测肝毒性标志物,并结合微核试验和单细胞凝胶电泳试验建立体外细胞毒性和遗传毒性的快速筛选平台。方法选取适当的荧光探针Hoechst33342、DCFH-DA、Fluo4-AM、Mito Tracker?Red CMX Ros联合高内涵技术研究不同大黄蒽醌类单体(AQs)对HepaRG细胞活性氧簇(ROS)、胞内Ca2+含量及线粒体膜完整性等肝毒性标志物的影响,并开展高内涵法胞质分裂阻断法微核试验和高通量彗星电泳试验,综合评价AQs致肝细胞毒性及染色体、DNA损伤情况。结果与对照组比较,HepaRG细胞经25.0μg/m L大黄素、12.5和25.0μg/m L芦荟大黄素、50和25.0μg/m L大黄酚处理24 h后,胞内ROS含量显著增多;12.5和25.0μg/m L芦荟大黄素和50.0μg/m L大黄酸可引起胞内Ca2+含量显著增多;大黄素25.0μg/m L、芦荟大黄素25.0μg/m L、大黄酚50.0和25.0μg/m L、大黄酸50.0和25.0μg/m L组导致线粒体明显损伤(P<0.05、0.01)。与对照组比较,25.0μg/m L大黄素诱导微核率、尾DNA含量和彗星尾距(OTM)数值均显著升高(P<0.05、0.01);50.0μg/m L大黄酚给药72 h后微核率显著升高(P<0.01)。结论 AQs的研究结果与现有文献报道基本相符。本研究成功建立肝细胞毒性和遗传毒性的联合快速筛选模型,有助于药物研发早期的毒性筛选。
Objective To establish a rapid screening platform for cytotoxicity and genotoxicity in vitro using HepaRG and high connotation technologies to detect hepatotoxicity markers in combination with micronucleus test and single cell gel electrophoresis assay. Methods The appropriate fluorescent probe Hoechst33342, DCFH-DA, Fluo4-AM, Mito Tracker? Red CMX Ros combined with high-content technology were used to study the effects of different anthraquinones (AQs) on reactive oxygen species (ROS) Ca2 + content and integrity of mitochondrial membrane and other hepatotoxicity markers, and carry out high connotation method of cytokinesis-blocking micronucleus test and high-throughput comet assay, comprehensive evaluation of AQs induced hepatotoxicity and chromosomes, DNA damage . Results Compared with the control group, the intracellular ROS content of HepaRG cells was significantly increased after treatment with 25.0 μg / mL of emodin, 12.5 and 25.0 μg / mL of aloe-emodin, 50 and 25.0 μg / mL of chrysophanol for 24 h, respectively. And 25.0 μg / m L aloe-emodin and 50.0 μg / m L rhein significantly increased intracellular Ca 2+ concentration; emodin 25.0 μg / m L, aloe-emodin 25.0 μg / m L, chrysophanol 50.0 and 25.0 μg / m L, rhein acid 50.0 and 25.0 μg / m L resulted in significant mitochondrial damage (P <0.05, 0.01). Compared with the control group, the micronucleus rate, the tail DNA content and the comet tail (OTM) value of 25.0μg / m L emodin were significantly increased (P <0.05,0.01); 50.0μg / m L chrysophanol administered 72 The micronucleus rate was significantly increased after h (P <0.01). Conclusions The results of AQs are in good agreement with the existing literature. In this study, we established a combined rapid screening model of hepatotoxicity and genotoxicity, which is helpful for the early toxicological screening of drug development.